Hashimoto K, Koga M, Motomura T, Kasayama S, Kouhara H, Ohnishi T, Arita N, Hayakawa T, Sato B, Kishimoto T
Department of Medicine III, Osaka University Medical School, Japan.
J Clin Endocrinol Metab. 1995 Oct;80(10):2933-9. doi: 10.1210/jcem.80.10.7559877.
The expression of GHRH receptor (GHRH-R) messenger ribonucleic acid (mRNA) was studied in 22 pituitary adenomas and 2 normal anterior pituitaries. Northern blot analysis revealed that GHRH-R mRNA were expressed in all 14 GH-producing adenomas, 1 of 3 ACTH-producing adenomas, the 1 PRL-producing adenoma, 2 of 4 nonfunctioning adenomas, and the 2 normal anterior pituitaries. Their expression levels varied among GH-producing adenomas and were relatively low in GH-nonproducing adenomas. In addition to the major transcript with a molecular mass of 2.0 kilobases (kb), the transcripts were identified at 2.8 and 4.5 kb in some GH-producing adenomas. To examine the structural variations in GHRH-R mRNA in pituitary adenomas, we amplified the complementary DNA fragment encompassing the region from the third cytoplasmic loop to the sixth transmembrane domain of GHRH-R. This region was selected because this region of the G protein-coupled receptor has been known to interact with G protein. Two amplified fragments with the molecular masses of 250 and 810 base pairs were identified by the reverse transcriptase-polymerase chain reaction method. The nucleotide sequence of a smaller fragment, which was the expected size, revealed that no mutations were found in this region in 10 GH-producing adenomas examined. However, a larger fragment contained the currently unidentified insertion. Compared with the genomic DNA sequence, this insertion was found to be generated through alternative splicing. In addition, this variant form contained the premature stop codon in-frame, indicating that it encodes the truncated GHRH-R. This insertion-specific probe could hybridize with 2.8- and 4.5-kb species of GHRH-R mRNA on Northern blot analysis, and these transcripts were expressed mainly in GH-producing adenomas. Finally, study of cell transfection and cAMP measurement revealed that this truncated GHRH-R was unable to transmit GHRH signals. These results suggest that some GH-producing adenomas preferentially express the truncated GHRH-R as a nonfunctioning receptor through alternative splicing.
在22例垂体腺瘤和2例正常垂体前叶中研究了生长激素释放激素受体（GHRH-R）信使核糖核酸（mRNA）的表达。Northern印迹分析显示，GHRH-R mRNA在所有14例生长激素分泌型腺瘤、3例促肾上腺皮质激素分泌型腺瘤中的1例、1例催乳素分泌型腺瘤、4例无功能腺瘤中的2例以及2例正常垂体前叶中均有表达。它们的表达水平在生长激素分泌型腺瘤中各不相同，在不分泌生长激素的腺瘤中相对较低。除了分子量为2.0千碱基（kb）的主要转录本外，在一些生长激素分泌型腺瘤中还鉴定出了2.8 kb和4.5 kb的转录本。为了研究垂体腺瘤中GHRH-R mRNA的结构变异，我们扩增了包含GHRH-R从第三个细胞质环到第六个跨膜结构域区域的互补DNA片段。选择该区域是因为已知G蛋白偶联受体的该区域与G蛋白相互作用。通过逆转录聚合酶链反应方法鉴定出两个分子量分别为250和810碱基对的扩增片段。较小片段的核苷酸序列（预期大小）显示，在所检测的10例生长激素分泌型腺瘤中，该区域未发现突变。然而，较大片段包含目前未知的插入序列。与基因组DNA序列相比，发现该插入序列是通过选择性剪接产生的。此外，这种变异形式包含框内过早终止密码子，表明它编码截短的GHRH-R。这种插入特异性探针在Northern印迹分析中可与2.8 kb和4.5 kb的GHRH-R mRNA杂交，这些转录本主要在生长激素分泌型腺瘤中表达。最后，细胞转染和cAMP测量研究表明，这种截短的GHRH-R无法传递GHRH信号。这些结果表明，一些生长激素分泌型腺瘤通过选择性剪接优先表达截短的GHRH-R作为无功能受体。
